Mitochondrial dynamics as a novel treatment strategy for triple‐negative breast cancer

Abstract Introduction Triple‐negative breast cancer (TNBC), recognized as the most heterogeneous type of breast cancer (BC), exhibits a worse prognosis than other subtypes. Mitochondria dynamics play a vital role as mediators in tumorigenesis by adjusting to the cell microenvironments. However, the relationship between mitochondrial dynamics and metabophenotype exhibits discrepancies and divergence across various research and BC models. Therefore, this study aims to explore the role of mitochondrial dynamics in TNBC drug resistance and tumorigenesis. Methods The Wst‐8 test was conducted to assess doxorubicin sensitivity in HCC38, MDA‐MB‐231 (TNBC), and MCF‐7 (luminal). Confocal microscopy and FACS were used to quantify the mitochondrial membrane potential (ΔφM), mitophagy, and reactive oxygen species (ROS) production. Agilent Seahorse XF Analyzer was utilized to measure metabolic characteristics. Dynamin‐related protein‐1 (DRP1), Parkin, and p62 immunohistochemistry staining were performed using samples from 107 primary patients with BC before and after neoadjuvant chemotherapy (NAC). Results MDA‐MB‐231, a TNBC cell line with reduced sensitivity to doxorubicin, reduced ΔφM, and enhanced mitophagy to maintain ROS production through oxidative phosphorylation (OXPHOS)‐based metabolism. HCC38, a doxorubicin‐sensitive cell line, exhibited no alterations in ΔφM or mitophagy. However, it demonstrated an increase in ROS production and glycolysis. Clinicopathological studies revealed that pretreatment (before NAC) expression of DRP1 was significant in TNBC, as was pretreatment expression of Parkin in the hormone receptor‐negative group. Furthermore, low p62 levels seem to be a risk factor for recurrence‐free survival. Conclusion Our findings indicated that the interplay between mitophagy, linked to a worse clinical prognosis, and OXPHOS metabolism promoted chemotherapy resistance in TNBC. Mitochondrial fission is prevalent in TNBC. These findings suggest that targeting the unique mitochondrial metabolism and dynamics in TNBC may offer a novel therapeutic strategy for patients with TNBC.

Breast cancer (BC) is the most frequently diagnosed malignant tumor among women globally and ranks as the second leading cause of mortality. 1Typically, it is classified based on hormone receptor (HR) expression, specifically including estrogen receptor (ER) and progesterone receptor (PR), as well as the presence of human epidermal growth factor receptor 2 (HER2).However, approximately 10%-20% of BC lacks expression of ER, PR, and HER-2, categorizing it as triple-negative breast cancer (TNBC), which is one of the most heterogeneous subtypes associated with an unfavorable prognosis. 2Some novel targets, such as immune checkpoint inhibitors, have shown improved response rates when used in combination with anthracycline and taxane-based chemotherapy.Nevertheless, early recurrence continues to significantly worsen patient prognosis. 3,4Therefore, TNBC urgently requires innovative and more potent treatment strategies.
The discovery of the Warburg effect, where cancer cells maintain aerobic glycolysis even in the presence of sufficient oxygen, has led to the widespread adoption of 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography (FDG-PET) combined with computed tomography for cancer detection. 5,6Owing to its quicker assessment of tumor size than conventional imaging and its sensitivity in indicating chemotherapy efficacy, such as evaluating neoadjuvant chemotherapy (NAC) response during BC treatment, FDG-PET is the preferred imaging method for assessing chemotherapy efficacy. 7][10][11] Studies have revealed the survival of patients with BC with minimal FDG uptake, indicating that BC cells utilize other metabolic pathways. 12lutamine metabolism, the pentose phosphate pathway, hexosamines, amino acids, and lipids have been observed to be closely related to oxidative phosphorylation (OXPHOS), providing support for cell proliferation. 9,13In our previous research, we revealed the impact of metabolic products of invasive ductal carcinoma on pathways involving thymidine, alanine, asparagine, glutamine, arginine, and proline. 9,14tochondrial dynamics, encompassing fusion, fission, and mitochondrial autophagy processes, constitute fundamental components of the tumor signaling pathway and are closely associated with mitochondrial metabolism, ensuring cellular adaptability. 15Studies have explored variations in the expression of mitochondrial dynamics within TNBC. 16,17However, the relationship between mitochondrial dynamics and metabophenotype exhibits discrepancies and divergence across various research and BC models. 17,18Mitophagy, a process involving the selective autophagy of mitochondria, maintains intracellular environment stability. 19he most well-established pathway is initiated by the phosphatase and tensin homolog (PTEN) and tension protein homolog (PINK1), leading to the activation of the putative kinase 1 (PINK1)/Parkin pathway.1][22][23] As the main drug in the classic formula for NAC treatment, doxorubicin is widely used and has remarkable effects, but it also has widespread drug resistance problems. 24,25In addition, we also got some inspirations on mitochondrial metabolism from the limitations of FDG-PET.We hypothesize that drug-resistant TNBC may mainly rely on mitochondrial metabolism and active mitochondrial dynamics (such as mitochondrial fission and mitophagy) to evade drug effects and FDG-PET tracking.Therefore, this study designs first using luminal, TNBC cell lines, and corresponding cell models under doxorubicin treatment for the aim of exploring mitochondrial metabolism and mitophagy and subsequently exploring the relationship between them and doxorubicin resistance in NAC through clinical pathology studies.

| Cell lines and culture
To demonstrate the specificity of TNBC among all subtypes, we selected the intracavitary cell line MCF-7, which exhibits a better prognosis and reduced sensitivity to chemotherapy, as the subtype control.TNBC cell lines, including MDA-MB-231, HCC38, and MCF-7, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Cosmo bio, USA) and 100 μg/mL penicillin/streptomycin (Gibco BRL, Grand Island, NY, USA) was used for cell culture.Cells were cultured under conditions of 37°C and 5% CO 2 .

| Cell proliferation assay
To identify cells with varying drug resistance serving as models for less effective and sensitive responses to doxorubicin, a cell viability test was conducted following 6 and 24 h doxorubicin treatments.The cell lines were uniformly seeded at a concentration of 4000 cells per well in a 96-well plate.Various concentrations of doxorubicin, ranging from 0.001 to 80 μM, were added to the culture medium, and the cells were incubated for 6 and 24 h.Cell viability was assessed using WST-8 (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan).Absorbance at 450 nm was measured using a cell counter (Sysmex CDA-500, Sysmex Corporation, Hyogo, Japan). 26

| Confocal microscope
ΔφM and reactive oxygen species (ROS) production were observed using confocal microscopy (Nikon Instruments A1 Confocal Laser Microscope Series With NIS-Elements C Software).This assessment was conducted considering mitochondrial depolarization as the initial mitochondrial response to drug-induced stress. 27he cells were placed in glass-bottom dishes with and without 100 nM doxorubicin and treated for 6 h.They were then stained with tetramethyl rhodamine, methyl ester (TMRM) (400 nM, Marker Gene Technologies INC, USA), and Mito Marker Green (MTG) (120 nM, Marker Gene Technologies, Inc, USA) for 30 min.Additionally, The CellROX ROS Detection Kit (1:1000, ab186029, Abcam) was incubated for 45 min in a cell culture incubator.Hoechst 33342 (1:10,000, H3570, Thermo Scientific, USA) was added and incubated for 10 min.The excitation wavelengths for TMRM, MTG, ROS, and Hoechst 33342 were 548, 490, 650, and 361 nm, respectively, while their emission wavelengths were 574, 516, 675, and 486 nm, respectively.

| Flow cytometry
ΔφM and mitochondria quantity within the cells were assessed using flow cytometry (BD FACS Aria™ III Cell Sorter).The cells were exposed to TMRM and MTG at concentrations of 400 nM and 120 nM, respectively, and incubated for 30 min.Following harvest through trypsinization and centrifugation (1000 rpm, 4°C, 5 min), the cells were set to a concentration of 1 million cells/mL using a staining buffer.Subsequently, 7-AAD (10 uL/mL, Bio Legend Way, San Diego) was introduced to ensure the living cells exceeded 85%, and the cell population was promptly assessed using flow cytometry.

| Seahorse XF mitochondrial stress assay
The Seahorse XFe96 Analyzer (Agilent, Santa Clara, USA) was used to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of cells.Cell plates containing 3 × 10 4 cells/well were seeded into Cell-Tak-coated 96-well Seahorse plates and pre-incubated at 37°C without CO2 for 30 min.OCR and ECAR were measured in XF media under basal conditions and in response to specific compounds (2 μM oligomycin, 2 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, 1 μM rotenone + antimycin A) using the Seahorse XFe96 Analyzer.

| Patient samples
This retrospective study utilized data from 107 consecutive patients with TNBC who underwent NAC and surgery at two institutions, Tohoku University Hospital and Tohoku Kosai Hospital, both located in Sendai, Japan, between 2015 and 2017.The specimens were obtained before (core needle biopsy) and after receiving NAC.The prognosis information for recurrence-free survival (RFS) of patients was last updated in January 2023.

| Assessment of immunoreactivity
Digital analysis software, "HALO TM Area Quantification ver.2.2 (Indica Labs, Corrales, NM)," was utilized to assess the distinct morphology of tumor cells and their immunoreactivity. 28This application initially separated IHC images into hematoxylin and DAB channels.Subsequently, it identified individual cells and graded them based on the intensity of the DAB signal in the cytoplasm.Results generated by HALO included counts and ratios of negative, high, medium, and low-intensity cells.The total score of IHC staining was determined by multiplying the intensity score (0 = negative, 1 = low, 2 = medium, and 3 = high intensity) by the proportion score, represented as a positive ratio ranging from 0 to 10 (corresponding to 0%-100%), yielding a final score between 0 and 30. 9We designated the antibody expression before NAC as "pre" and that after NAC as "post" to differentiate between the characteristics of expression before and after NAC.

| Data analysis
All experiments were performed at least thrice.ImageJ was used to digitize the immunofluorescence intensity of confocal microscopy and western blot.The Shapiro-Wilk test analyzed repeated experimental results and the data conformed to a normal distribution.Unpaired t-test was used to examine the changes in fluorescence intensity of cell lines treated with or without chemotherapy.Ordinary one-way ANOVA tested the differences of the three cell lines in Seahorse XF mitochondrial stress assay and western blot.Kolmogorov-Smirnov test demonstrated the normal distribution of clinical pathology analysis results.The chi-square test was used to determine the disparity in protein expression regarding clinical characteristics.Additionally, Cox univariate and multivariate analyses of variables were performed to assess the variables associated with RFS.The closer the Hazard ratio value is to 0, the lower the risk that the former variable of a pair of variables may cause RFS, and the higher the risk that the latter variable may cause RFS.Statistical significance was set at p < 0.05.The definition of statistical significance is as follows: p <*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Data analyses were conducted employing SPSS V.25 statistical software (Chicago, Illinois, USA) and GraphPad Prism version 9.5.0 for macOS (Boston, Massachusetts USA).

| MDA-MB-231 maintained the stability of ROS with doxorubicin
Subsequently, we assessed ROS production in TNBC cell lines, as superoxide accumulation is one of the mechanisms underlying the anticancer effects of chemotherapy medications. 29ROS production significantly increased in HCC38; however, it showed a minimal change in MDA-MB-231 after doxorubicin treatment (Figure 3A,B).The results showed that HCC 38 exhibited an increase in ROS levels, whereas MDA-MB-231 maintained a consistent ROS level after treatment with doxorubicin.(Figure 3C) (p = 0.0350, p > 0.05).The baseline ROS level in MDA-MB-231 was higher than that in HCC38.This suggests that MDA-MB-231 can tolerate cell stress changes for a longer duration and engage in more active signal conduction activities to promote cell proliferation and invasion (Figure 3C) (p = 0.0022). 30

| MDA-MB-231 utilized mitophagy to maintain ROS stability
As the reduced ΔφM is considered to contribute to mitochondrial autophagy, we conducted flow cytometry to assess the mitochondrial quantity.The MTG curve of HCC38 showed minimal changes after treatment with doxorubicin (Figure 4A, p > 0.05).In contrast, MDA-MB-231 treated with doxorubicin exhibited an increase than the control group, indicating a decrease in mitochondrial content following doxorubicin treatment (Figure 4B, p = 0.00691).
The decreased ΔφM and mitochondrial content in MDA-MB-231, which were less responsive to the drug, indicates the presence of mitophagy.After mitophagy, P62 binds to LC3B to form autophagosomes and degrades, while LC3B dissociates. 31Therefore, we examined mitophagy-related proteins, specifically LC3B and p62.The results showed that p62 exhibited high expression in TNBC cell lines but was scarcely expressed in MCF7.Furthermore, p62 expression in MDA-MB-231 decreased significantly than in HCC38 following exposure to doxorubicin (Figure 4C,D, p = 0.0009).LC3B exhibited significantly high expression levels exclusively in MDA-MB-231 (Figure 4E).After doxorubicin treatment, LC3B expression increased slightly (n.s) (Figure 4F).These findings suggest that MDA-MB-231 specifically utilizes mitochondrial autophagy to alleviate excessive ROS pressure, potentially leading to drug resistance.

| Less effective cell line doxorubicin relies on OXPHOS metabolism, whereas doxorubicin-sensitive cell lines depend on glycolysis
MDA-MB-231, a cell line with poor responsiveness to doxorubicin, was found to maintain stable levels of ROS through mitochondrial autophagy.Considering the correlation between these mechanisms and cellular metabolic phenotypes, we hypothesized that these cell lines would exhibit distinct metabolic characteristics. 32,33The Seahorse XFe96 Analyzer was used to measure oxidative phosphorylation (OXPHOS) through OCR and glycolysis through ECAR (Figure 5A). 34Spare respiratory capacity (SRC) represents an indicator of cellular responsiveness to energy demands or cellular health.Figure 5A displays additional metrics for better understanding.
The results showed that the initial ECAR of HCC38 was significantly higher than that of the other two cell lines (Figure 5B,C, p < 0.001).Conversely, the base OCR of MDA-MB-231 and MCF-7 exceeded that of HCC38 (Figure 5D,E, p = 0.0162).The SRC of HCC38 was significantly lower than that of others (p < 0.005) (Figure 5F).Doxorubicin treatment had no significant effect on metabolic changes in all cell lines.Therefore, considering the changes in ΔφM, mitophagy conditions, and metabolism features, HCC38 exhibited sensitivity to doxorubicin primarily through glycolysis, while doxorubicin-less effective cells, MDA-MB-231, and MCF-7 mainly use OXPHOS for metabolism.These findings reveal mitophagy combined with OSPHOS metabophenotype to mitigate excess ROS stress.This phenomenon may have contributed to NAC resistance specifically observed in TNBC.

| Mitophagy-related proteins were expressed in TNBC
The study has demonstrated a unique coexistence of mitophagy and OXPHOS as a mechanism that enables drug resistance to external pressure in TNBC cell lines, which were less responsive to treatment.Therefore, clinicopathological studies were conducted to examine the relationship between mitochondrial dynamic features and in vitro clinical aspects.DRP1 induces mitochondrial fission while Parkin, a component of the PINK1/Parkin-mediated pathway, functions as a tumor suppressor.P62 is packaged with mitochondria into mitolysosomes. 35Therefore, these three proteins were assessed in 107 patients.
Seventy-seven patients (78%) had lymph node metastasis, and 96 patients (86.7%) had received anthracyclines (Table 1).Thirty-one patients (29%) achieved pathological complete response (Grade 3, pCR) after NAC (Table 2).FDG-PET is a potent imaging tool employed for assessing tumor glycolysis and FDG uptake, which is quantified as the maximum standardized uptake value (SUVmax).This technique is commonly utilized to assess cancer aggressiveness. 36Among patients who underwent FDG-PET after NAC and had a post-SUV max value <2.5, there was a notably high non-pCR rate of 60.5%.This suggests that FDG-PET assessment after NAC may not be a reliable predictor of pCR (Table 2).Figure 6A shows the representative images of DRP1, p62, and Parkin expression.Figure 6B shows the IHC images of four intensity scores.We categorized the total scores into high and lowexpression groups, using a cutoff of 10.5.The results indicated that pre-DRP1 exhibited high expression levels in Abbreviations: ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; pCR, pathological complete response; post, after chemotherapy; pre, before chemotherapy; SUVmax, maximum standardized uptake value; TNBC, triple negative.specific patient groups, including those under 50 years of age (<50), individuals with ER-/PR-(HR-) status, patients with TNBC, and those with a high Ki-67 (20% cutoff) rate (p = 0.002, 0.031, 0.013, 0.015, respectively) (Table 3).No correlation was observed between pre-/post-DRP1 expression and the NAC effect (Tables 3 and 4).Pre-Parkin was also expressed in HR-(Table 3, p = 0.034).We performed a multivariable analysis on factors that demonstrated pvalues below 0.2 in the univariable analysis.Pre-DRP1 and pre-Parkin were not risk factors for RFS in patients with breast cancer (Figure 6C,D).Low expression of pre-P62 showed a higher risk of RFS than high pre-p62 expression, although this was not significant (Figure 6E; Table 5) (HR = 2.459, p = 0.082).

| DISCUSSION
Despite significant progress in breast cancer screening, diagnosis, and treatment, TNBC remains a formidable challenge for clinical oncologists.This is primarily attributed to the unfavorable patient outcomes associated with TNBC.Conventional therapeutic approaches, including endocrine and anti-HER2 therapies, which are often effective in treating other types of BC, proved ineffective in TNBC owing to the absence of hormone receptors and HER2 amplification.Therefore, chemotherapy is the primary therapeutic approach for TNBC treatment.NAC is a proven treatment for high-risk, locally advanced, or unresectable breast cancers that allows the evaluation of treatment response and enables the discontinuation of treatment in cases with tumor progression.Moreover, pCR is used as a surrogate marker for disease-free survival and overall survival. 37Among patients with breast cancer treated with NAC, TNBC has the most favorable pCR rate compared to other subtypes. 38Furthermore, previous studies have shown that the pCR rate in TNBC is most significantly correlated with its prognosis.Consequently, it is essential to investigate sensitivity to NAC treatment. 39rogrammed death ligand 1 (PD-L1) is gaining wide interest in TNBC, with a positivity rate of 20%-60%.However, approximately 30% of patients with TNBC still achieve non-pCR, with a significant probability of recurrence. 3,40ocusing on mitochondrial metabolic signatures associated with tumors could offer additional therapeutic options for this aggressive BC subtype.This is particularly relevant because metabolism is recognized as one of the hallmarks of cancer.However, metabolic-based therapy, other than the progress in the treatment of glycolytic subtypes, provides less information. 41n our study, we found through cell experiments that the less effective TNBC cell line MDA-MB-231-specific mitophagy combined with OXPHOS metabolism resisted the therapeutic effect of doxorubicin.This result has also been shown in clinical pathological analysis and has certain significance which firstly demonstrated the unique contribution of high mitochondrial metabolism with mitophagy to TNBC drug resistance from the perspective of T A B L E 4 Clinicopathological features and association with the status of post-DRP1, p62, and Parkin.FDG-PET limitations and pilot studies of mitochondrial metabolites.We also suggested the significance of detecting and evaluating mitochondrial metabolism, mitochondrial fission in TNBC patients and mitophagy related to worse prognosis, provided theoretical support for the next study of OSPHOS-tracer-PET/mitochondrial autophagy tracer-PET.Doxorubicin induces iron chelation, leading to a rapid increase in ROS levels and thus accelerating death of cancer cells. 42The rapid increase of ROS in HCC38 and the stable maintenance of ROS in MDA-MB-231 suggest that HCC38 is sensitive and MDA-MB-231 is less effective to doxorubicin.Metabolic analysis revealed that doxorubicin-less effective cell lines are dependent on OXPHOS.Another study also found that after glycolysis is inhibited, glucose metabolism is transferred to OXPHOS in an autophagy-dependent manner, which promotes cell survival, confirming our results. 43Chemotherapy had little effect on metabolic status; however, combined with intracellular ROS production, we observed ΔφM, mitochondrial loss, and specific expression of LC3B and p62, which indicated that the coexistence of mitophagy and OXPHOS avoids drug intervention only in the doxorubicinresistant TNBC cell line.The resistance mechanism of the luminal cell line is independent of mitophagy.

Post-DRP1
Subsequently, we performed an IHC analysis to investigate the potential association between mitochondrial fission, canonical mitophagy, and drug sensitivity.The limitations of FDG-PET in predicting pCR highlight its inability to assess non-glycolytic metabolism in BC.DRP1 is highly expressed in TNBC, indicating mitochondrial fission and low p62 expression seems to be a high-risk factor for RFS, indicating mitophagy with a poor prognosis.DRP1 regulates mitochondrial fission, which plays a crucial role in the initiation of mitophagy.This process encompasses both canonical and receptor-mediated pathways. 35Receptor-mediated mitophagy, which involves proteins such as FUNDC1, BBNIP3, and BNIP3L/NIX, is associated with treatment in cancer cells and prognosis.Mitophagy controlled by BNIP3L pathways protects glioblastoma cells from lack of oxygen. 44,45herefore, the overexpression of DRP1 induces mitochondrial fission, leading to a decrease in ΔφM levels when mitochondria are under stress, thereby initiating mitochondrial autophagy.In the context of fragmented and damaged mitochondria, phosphatase and tensin homolog-induced kinase 1 (PINK1) recruits Parkin.Subsequently, the adaptor protein p62 recognizes phosphorylated polyubiquitin chains and initiates the formation of the mitochondrial autophagosome by binding to microtubule-associated protein LC3. 35The classical pathway of PINK1-Parkin-mediated mitochondrial autophagy is named after juvenile Parkinson's disease.It is noteworthy that Parkin often functions as a tumor suppressor. 46,47The upregulation of PINK1 in lung and esophageal cancers indicates classical mitochondrial autophagy associated with chemotherapy resistance. 47,48Both mitochondrial autophagy pathways are implicated in the therapeutic resistance of hepatocellular carcinoma. 20,43These findings align with the phenomenon of mitochondrial autophagy observed in TNBC cells.Our study underscores oxidative phosphorylation as a primary metabolic signature and elucidates the role of mitochondrial autophagy in cancer development.
Our study had some limitations.First, our understanding of the impact of mitophagy on breast cancer, including both the canonical and other mitophagy pathways, remains insufficient.In the future, it will be essential to conduct more experiments using mitophagy-related gene knockout and amplification cell models.These experiments will assist in identifying targets for monitoring mitophagy, mitochondrial metabolism, and potential therapies.In addition, We have not yet identified suitable targets for PET or other imaging and non-invasive detection methods in patients with mitochondrial metabolism.This remains one of our future research objectives.If feasible, we will also identify the medications required to alter the metabolic state when mitophagy or a prolonged OXPHOS metabolic state is present.Finally, in pathological clinical analysis, it is challenging to eliminate the influence of different kinds of drugs used in NAC regimens on the expression of these proteins.Exploring whether mitophagy is involved in the response of tumors to other drugs remains a subject that requires ongoing investigation.

| CONCLUSIONS
TNBC cells possess the capacity for efficient mitochondrial autophagy while relying on oxidative phosphorylation for metabolism, enabling resistance to and survival during chemotherapy.In contrast, cells dependent on glycolysis are unable to initiate mitochondrial autophagy to counteract the high levels of ROS generated by chemotherapy, ultimately leading to cell death.Collectively, we have demonstrated a high occurrence of mitochondrial autophagy in TNBC cell lines, which is associated with an unfavorable prognosis in breast cancer.In the future, we hope to conduct in-depth studies to investigate multiple mitophagy pathways.This is necessary to explore novel therapeutic options that target the unique mitochondria metabolic signatures within tumor cells.

F
I G U R E 1 Cell viability assay and IC50.(A) IC50 of doxorubicin in different cell lines.(B) (C) Cell viability assay by using doxorubicin on three cell lines (MDA-MB-231, HCC38, and MCF-7) in 6 and 24 h.

F I G U R E 2
ΔφM of HCC38, MDA-MB-231, and MCF7.(A, B) Confocal microscopy images at 100× magnification were captured for HCC38 cells, focusing on ΔφM (control and doxorubicin).Red fluorescence (TMRM) represents ΔφM.MTG was used as a mitochondrial localizer (green).Nuclei were co-stained using Hoechst 33342 (blue).(B) Flow cytometry analysis of TMRM fluorescence in HCC38 cells (control and doxorubicin-treated). (C, D) We obtained 100× confocal microscopy images targeting TMRM and performed flow cytometry analysis to measure TMRM fluorescence in MDA-MB-231 (control and doxorubicin-treated). (E, F) We captured 100× confocal microscopy images targeting TMRM and performed flow cytometry analysis to measure TMRM fluorescence in MCF-7 cells (control and doxorubicintreated).An unpaired t-test was used to ensure the consistency of repeated experimental results.

F
I G U R E 3 MDA-MB-231 maintained the stability of ROS after doxorubicin treatment.(A, B) Images of ROS fluorescence were obtained from HCC38 and MDA-MB-231 cells (control and doxorubicin-treated) under 20× and 100× magnification.Red fluorescence (Ros deep red kit) represents ROS production.Nuclei were co-stained by Hoechst 33342 (blue).The 20× image captures a large number of cells, reflecting changes in overall ROS production, and the 100× magnification captures representative cells.(C) A histogram was created to display the mean intensity of fluorescence in MDA-MB-231 and HCC38 cells, with HCC38 (control) as the denominator using the lowest value as the reference point.An unpaired t-test was used to ensure the consistency of repeated experimental results.F I G U R E 4 MDA-MB-231 specifically used mitophagy to maintain ROS stabilization.(A, B) In flow cytometry, we measured MTG fluorescence in HCC38 and MDA_MB-231 cells (control and doxorubicin).(C) p62 expression in MDA-MB-231, HCC38, and MCF-7 (control and doxorubicin) by western blotting.(D) Quantification and comparison of p62 expression in three cell lines (control and doxorubicin).(E) LC3B expression in MDA-MB-231, HCC38, and MCF-7 (control and doxorubicin) by western blotting.(F) Quantification of western blots in E. Ordinary one-way ANOVA was used to ensure the consistency of repeated experimental results.F I G U R E 5 The cell line less responsive to doxorubicin primarily relies on OXPHOS metabolism, in contrast to HCC38.(A) Interpretation of the components of the Seahorse XF cell mitochondrial stress assay.(B, C) ECAR-glycolysis and OCR-mitochondrial respiration (OXPHOS) in three cell lines, both with (+) and without (−) doxorubicin injection.(D) Histogram of basal ECAR in three cell lines.Basal ECAR of HCC38 is the highest (p < 0.0001).(E) Histogram of basal OCR in three cell lines.The basal OCR of HCC38 is the lowest (p = 0.0162).(F) Histogram of SRC in three cell lines.SRC of HCC38 is the lowest (p = 0.0034).An ordinary one-way ANOVA was used to ensure the consistency of repeated experimental results.

T A B L E 2
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Comparison of assessments between pathologic response and FDG-PET.F I G U R E 6 Mitophagy-related proteins are highly expressed in TNBC patients.(A) IHC images of positive controls of DRP1 (kidney), Parkin (gall bladder), and p62 (HCC).(B) IHC images of four intensity scores.(C-E) RFS with pre-DRP1, pre-p62, and pre-Parkin expression.The data were analyzed using the chi-square test and Cox regression analysis.
Clinicopathological features and association with the status of pre-DRP1, p62, and Parkin.